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The present invention relates to increasing bacterial toxin production using methods and compositions that reduce, or eliminate, the accumulation of intracellular and extracellular toxin expression inhibitors. Specifically, the present invention related to methods and compositions for reducing or elimination the accumulation of Bordetella species toxin expression inhibitors. More specifically, the present invention relates to the high yield production of pertussis toxin, pertactin, adenylate cyclase toxin-hemolysin, filamentous hemagglutinin and other toxins.
Pertussis toxin (PT) is one of the various components produced by virulent B. pertussis, the microorganism that causes whooping cough. Whooping cough is a serious infection of the respiratory system that at one time was responsible for the death of 5,000 to 10,000 people in the United States each year. Since the advent of the whooping cough vaccine the number of whooping cough related deaths has been reduced to less than 20 annually. Currently, about 50% of all whooping cough infections occur in children less than 1 year old, and only 15% occur in children over than 15 years old. Kids Health.org (visited Mar. 23, 2000)  less than http://kidshealth.org/parent/common/whooping_cough.html greater than .
PT is a major protective antigen in the vaccine against whooping cough. Other components of interest produced by B. pertussis are filamentous hemagglutinin, heat labile toxin, adenylate cyclase and the like, which may also play important role as protective antigens. Large-scale production of these components, which are useful as diagnostic or chemical reagents and in the preparation of vaccines, requires large-scale cultivation of the microorganism. However, B. pertussis is a fastidious organism that has proved difficult to grow in large fermentors. Older methods for the culture of B. pertussis employ cultivation in stationary culture or in fermentors. Growth in a stationary culture is labor intensive, while cultivation on a fermentation scale requires vortex stirring and surface aeration. As a result, the effective volume of the fermentor is reduced and modification of the fermentor for growth of pertussis is often necessary. Furthermore, the quantities of PT produced during fermentation under these conditions are variable and often low.
U.S. Pat. No. 5,338,670 discloses a method for the production of B. pertussis in the presence of an iron salt, namely ferrous sulfate. While high iron content supports greater bacterial growth, it suppresses the production of PT. By adjusting the iron content of modified Stainer-Scholte media to 10% of the recommended concentration, the production of PT was optimized.
The present invention seeks to improve the yield of PT obtained from B. Pertussis by (1) introducing a soluble salt into the growth medium that sequesters sulfate (SO42xe2x88x92) and/or (2) employing a B. Pertussis cysteine desulfinase knockout mutant.
The present invention is based upon the discovery that bacterial toxin expression inhibitors accumulate in culture media and thus significantly reduce toxin production. Moreover, the present invention is based on the findings that suppressing or eliminating toxin expression inhibitors can significantly up regulate toxin expression. Non-limiting examples of the present invention are disclosed using Bordetella sp., specifically, B. Pertussis and/or B. bronchiseptica which produce pertussis toxin (PT) and pertactin respectively. However, it is understood, that higher bacterial toxin levels can be achieved in other bacterial culture systems using the teachings of the present invention including but not limited to adenylate cyclase toxin-hemolysin, and filamentous hemagglutinin.
Generally, the present invention is exemplified by disclosing methods and compositions used to cultivate B. Pertussis that eliminate, or reduce, intracellular and extracellular PT inhibitor accumulation resulting in significant PT production increases.
In one embodiment of the present invention methods and compositions for preparing novel culture media that support B. Pertussis growth and prevent or decrease PT inhibition expression by sulfate anions are disclosed. These media compositions and related methods include, but are not limited to, admixing a B. Pertussis culture medium with an effective amount of one or more soluble metal salts that form substantially insoluble complexes with sulfate anions.
In another embodiment of the present invention culture media that support B. pertussis growth comprising an amount of one or more soluble salts that form substantially insoluble complexes with PT inhibitors, wherein said amount prevents or reduces the inhibition of PT expression are provided. Specifically, soluble metal salts are disclosed that form substantially insoluble complexes with sulfate anions.
Other embodiments of the present invention include B. Pertussis culture media and methods for making and using same that reduce PT inhibitors by limiting or eliminating media constituents that contribute to PT inhibitor accumulation. Specifically, in one embodiment of the present invention cysteine concentration is reduced.
The invention also relates to methods and compositions for producing PT comprising cultivating B. Pertussis under conditions that eliminate, or reduce, the accumulation of PT inhibitors in the culture media resulting in significant PT production increases and isolating the PT from the culture medium.
In yet another embodiment of the present invention PT production is enhanced using B. Pertussis cysteine desulfinase knockout mutants. In one embodiment of the present invention methods of producing PT comprising growing a B. Pertussis cysteine desulfinase knockout mutant in a B. Pertussis culture medium, and isolating the PT from the culture medium are provided.